Electronic-property probing of biological molecules at surfaces

Details Electronic-property probing of biological molecules at surfaces Electronic-property probing of biological molecules at surfaces

1997-04-10

2001-10-23

Houtteman, Scott W. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C536S024300, C536S026600

Reexamination Certificate

active

06306584

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the derivatization of surfaces for determination of analytes, for example from a fluid medium using a biological binding partner of an analyte, and more particularly to the formation, on a surface, of a self-assembled molecular monolayer including a biological species such as a nucleic acid strand, and the use of the self-assembled monolayer in a probe that acts to detect molecular recognition via electronic properties of the biological species.
BACKGROUND OF THE INVENTION
Biochemical analyses are invaluable, routine tools in health-related fields such as immunology, pharmacology, gene therapy, and the like. In order to successfully implement therapeutic control of biological processes, it is imperative that an understanding of biological binding between various species is gained. Indeed, an understanding of biological binding between various species is important for many varied fields of science.
Many biochemical analytical methods involve immobilization of a biological binding partner of a biological molecule on a surface, exposure of the surface to a medium suspected of containing the molecule, and determination of the existence or extent of molecule coupling to the surface-immobilized binding partner.
The study of biological binding involving nucleic acid at surfaces has been hindered by the difficulty in immobilizing a single strand of nucleic acid at a surface without also immobilizing the complement of that strand. Where a single strand of nucleic acid is immobilized at a surface with its complement, it is not available for interaction by itself.
Electron transfer through model enzymes has been studied, and several theoretical models predict rates of transfer through these enzymes (Chidsey, C.E.D., “Free Energy and Temperature Dependence of Electron Transfer at the Metalelectrolyte Interface,”
Science
251 (1991), pp. 919-922). Comparison of predicted electron transfer rates with the time required for electrons to travel a finite distance within a protein has led to the conclusion that electrons traverse a pathway of chemical bonds such as covalent or hydrogen bonds (J. N. Onuchic, D. N. Beratan, J. R. Winkler, and H. B. Gray,
Ann. Rev. Biophys. Biomol. Struct
., 21 349 (1992); D. N. Baratan, J. N. Onuchic, J. R. Winkler and H. B. Gray,
Science
, 258 1740 (1992); J. J. Regan, S. M. Risser, D. N. Beratan, and J. N. Onuchic,
J. Phys. Chem
., 97 13083 (1993)), but do not travel through vacant space (S. M. Risser, D. N. Beratan, and T. J. Meade,
J. Am. Chem. Soc
., 115 2508 (1993)). This finding was later modified to include electron transfer between &pgr;-stacked electron systems (F. Barigelletti, L. Flamnigni, V. Balzani, J. P. Collin, J. P. Sauvage, A. Sour, E. C. Constable, and A. C. M. W. Thompson,
J. Am. Chem. Soc
., 116 7692 (1994); J. N. Onuchic and D. N. Beratan,
J. Am. Chem. Soc
., 109 6771 (1987)). Subsequently, several groups measured rates of electron transfer through electroactive proteins (enzymes) using modified or unmodified electrodes, then microelectrodes (Hill, H. A. O., Klein, N. P., Murthy, A. S. N., Psalti, I. S. N.,
Chemical Sensors and Instrumentation
, (1989) pp. 105-113; Armstrong, F. A., Bond, A. M., Hill, H. A. O., Psalti, I. S. N., Zoski, C. G.,
J. Phys. Chem
. 93, (1989) pp. 6485-6493).
One drawback in these studies is that direct adsorption of protein onto an electrode typically resulted in loss of conductivity, presumably due to protein denaturation. A hydrophilic molecule (promoter) therefore was adsorbed to an electrode prior to adsorption of the electroactive protein in some cases. The promoter layer is designed to bind the protein of interest through hydrogen bonds, giving the electrons a suitable pathway. Notably, in these studies, electrons were observed to travel through an inert molecule, then through the electroactive molecule (Hill, H. A. O. and Lawrence, G. A., “Some Consequences of Mixed and Dilute Surface Modification of Gold Electrodes for Protein Electrochemistry,”
J. Electroanal. Chem
. 270 (1989) pp. 309-318). The amplitude of the signal was dependent upon the potential difference.
Rates of electron transfer have also been measured through DNA (C. J. Murphy, M. R. Arkin, Y. Jenkins, N. D. Ghatlia, S. H. Bossman, N. J. Turro and J. K. Barton,
Science
, 262 1025 (1993)). It has been shown that the rate of electron transfer through double-stranded DNA is much faster than through single-stranded DNA (T. J. Meade and Jon F. Kayyem,
Angew. Chem. Int. Ed. Engl
., 34 3, pp. 352-354 (1995), “Electron Transfer through DNA: Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors”).
Co-pending, commonly-owned U.S. patent application Ser. No. 08/312,388, filed Sep. 26, 1994 by Bamdad, et al., describes a technique for immobilization of single-stranded DNA at a surface as part of a self-assembled monolayer, and use of the arrangement in determination of biological binding partners of the DNA via Surface Plasmon Resonance (SPR), a technique that measures the very slight changes in mass that occurs at a surface upon biological binding of a binding partner to the surface-immobilized species.
Accordingly, it is an object of the invention to provide techniques for studying molecular interactions at surfaces.
SUMMARY OF THE INVENTION
The foregoing and other objects and advantages of the invention are achieved by providing a molecule having a formula X—R—Ch, in which X represents a functional group that adheres to surface such as a gold surface, R represents a spacer moiety that promotes formation of a self-assembled monolayer of a plurality of the molecules, and Ch represents a bidentate, tridentate, or quadradentate chelating agent that coordinates a metal ion. The chelating agent includes a chelating moiety and a non-chelating linker moiety, such that it can be covalently linked via its linker moiety to the spacer moiety while allowing the chelating moiety to coordinate a metal ion. According to a preferred aspect of the invention a metal ion is coordinated to the chelating agent, and a binding partner of a target molecule is coordinated to the metal ion. This arrangement is facilitated by selecting the chelating agent in conjunction with the metal ion such that the chelating agent coordinates the metal ion without completely filling the ion's coordination sites, allowing the binding partner to coordinate the metal ion via coordination sites not filled by the chelating agent. According to one aspect of the invention the binding partner is a biological species that includes a polyamino acid tag, such as a tag made up of at least two histidine residues, that coordinates the metal ion. In this context the term “adhere” means to chemisorb in the manner in which, for example, alkyl thiols chemisorb to gold.
The present invention also provides a species having a formula X—R—Ch—M—BP—BMol, in which X represents a functional group that adheres to a surface, R represents self-assembled monolayer-promoting spacer moiety, Ch represents a chelating agent that coordinates a metal ion, M represents a metal ion coordinated by the chelating agent, BP represents a biological binding partner of a biological molecule, and BMol represents the biological molecule. The binding partner is coordinated to the metal ion.
The invention also provides an article including a solid phase that has a surface. A plurality of chelating agents are immobilized at the surface in such a way that essentially each of the chelating agents is oriented so as that the chelating moiety of the agent, that is the electron donating portions of the agent, face in a direction away from the surface and is unencumbered by species, such as other chelating agents, that would interfere with the chelating function. This can be accomplished by isolating the chelating agent at the surface by non-chelating species. In this way each chelating agent can coordinate a metal ion so as to expose in a direction away from the surface at least two free metal coordination sites. According to one aspect of the

Affiliated with

Also associated with

Rating

Electronic-property probing of biological molecules at surfaces does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Electronic-property probing of biological molecules at surfaces, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Electronic-property probing of biological molecules at surfaces will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2616682